본 연구는 벼에서 양성자 펌프의 활성을 증가시키는 것으로 알려진 오옥실 결합단백질 ABP57 유전자의 과발현에 따른 분자생물학적 특성을 분석해 작물 분자육종을 위한 기초자료로 활용하고자 수행하였다.ABP57 유전자 과발현 형질전환체와 대비 품종 낙동벼의 뿌리조직에서 유전자 발현 변화를 비교 분석한 결과는 다음과 같다. 두 식물체간에 발현량이 2배 이상 차이를 보인 유전자 수는 29,389개 중 총 148개였으며, 그 가운데 65개는 발현량 증가를 보였다. ABP57 유전자 과발현 형질전환체에서 발현량 변화를 보인 유전자들의 생물적 기능을 분석하기 위해 유전자 온톨로지 분석을 수행한 결과 각 부문별로 생물적 작용 부문에 67개 세부항, 세포 구성요소 부문에 59개 세부항, 분자기능 부문에 29개 세부항에 각각 영향을 미치는 것으로 나타났다. ABP57 유전자 과발현에 따라 세 개 부문 모두에서 공통적으로 나타난 영향은 H+-ATPases의 활성 증가, 여기에 필요한 에너지원 관련 화합물(ATPs, purines) 생합성 및 대사관련 활동 증가, 이온의 막투과 및 운반체 활동 증가 등이 확인되어 Kim et al.(2001)의 선행 연구결과와 일치하는 경향이었다. 그러나, 엽록체의 일부 구성요소나 G-protein 결합 단백질 신호전달경로, cytokine 신호전달 경로 등과 관련된 단백질 활성 감소는 식물 생육에 부정적 영향을 미칠 것으로 추정된다.따라서, 향후 작물의 효율적 분자육종을 위해서는 개별 유전자의 명확한 기능 분석과 더불어 유전자 상호작용에 대한 네트워크 분석 등에 대한 연구개발 노력과 함께 형질전환 식물체에 대한 생리적 특성, 표현형, 농업형질 특성 등에 대한 효과적 분석을 위해 전통육종과의 지속적 교류와 협력이 반드시 필요할 것이다.

Bacillus thuringiensis (Bt) crystal protein genes encode insecticidal δ-endotoxins that are widely used for the development of insect-resistant crops. Common soybean is a crop of economic and nutritious importance in many parts of the world. Korea soybean variety Kwangan was transformed with Bacillus thuringiensis (Bt) crystal protein genes. These genes were transformed into Kwangan using highly efficient soybean transformation system. Transgenic plants with Bt crystal protein genes were confirmed for gene introduction and their expression using PCR, real-time PCR and RT-PCR. Currently, the confirmation of stable gene introduction with Bt genes is also performing by southern blot analysis and physiology test and agronomic characters are investigating.

Cry1Ac protein is known as one of toxin crystal proteins synthesized from Bacillus thuringenesis that plays a critical role for the insect resistance. Recently, cry1Ac genes have introduced into many plants in general and soybean as well. However, the gene expression of cry1Ac genes in transgenic plants remains low that need to be improved. Several mutations we reintroduced into the cry1Ac genes in order to enhance the insecticidal effect. In this study, the cry1Ac with mutant #2, #11 and #16 were transformed into Kwangan, a Korean soybean variety by using the “half-seed” method. The plant lets carrying modified cry1Ac genes were primarily selected on media containing Phosphinothricine (PPT), a bar selective agent and Basta leaf painting. Then, the presence of introduced genes in T0 plants and the gene expression were investigated by PCR, RT-PCR and Real-time PCR. PCR and RT-PCR analysis showed expression of bar and cry1Ac genes from tested transgenic soybean plants. The number of copy of bar gene ranged from 1 to 3 by Real-time PCR analysis. These results provided a fundamental back ground for our further experiments: Confirmation of the gene expression by Southern blot and identification of the function of modified cry1Ac by insect bioessays.

In plants, the Dof (DNA binding with One Finger) proteins are plant-specific transcription factors with a particular class of zinc-finger DNA-binding domain. The Dof genes have been predicted 30 different Dof genes in the rice Oryza sativa genome by phylogenetic analysis. The mostly Dof proteins contain a conserved region of 50 amino acids with a C2-C2 zinc finger motifs that binds a cis-regulatory element sequence 5’-T/AAAAG-3’. We found that a member of the DOF transcription factor family, Dof1 gene of rice, was expressed to wound from Ds insertion mutant population. Sequencing of the flanking regions of the transposon insertion site indicated that the gene-trap had been inserted near the front of the second exon of OsDof1 gene in chromosome 7. Genomic southern analysis revealed that mutant line contained a single copy of Ds gene trap. The Ds tagged rice mutant line, OsDof1::Ds, wound-inducible GUS expression was identified. To analyze the cis-acting elements, we constructed fusion genes with the OsDof1 promoter fused to the β-glucuronidase (GUS) reporter gene and transformed Arabidopsis and rice plants with these constructs. Wound-induced GUS expression was observed in the leaves of transgenic OsDof1::GUS rice and Arabidospsis plants. These results showed that, OsDof1 protein might be involved in stress responses and growth regulation in plant, might plays a role as a transcription regulator in stress response signal transduction pathways of plant.

Transgenic cymbidium plants containing drought and salt stresses tolerance genes were produced by using a highly efficient Agrobacterium-mediated transformation system. The gene (AtSZF2) is salt and drought stresses tolerant gene and transferred into cymbidium plants. These transgenic cymbidium plants are investigated for gene introduction by PCR and analyzed by salt and drought stresses to check its gene expression. To investigate the gene expression of AtSZF2, leaves of transgenic cymbidium plants were soaked in salt solution (200 mM NaCl). Also, transgenic cymbidium plants were kept under no watering for 6 weeks to check the expression of drought stress tolerance. As a result, wild type plants showed more damage than transgenic plants under salt treatment. Further, transgenic cymbidium plants retained green color and healthy status, while wild type plants showed no tolerance after 6 weeks of no-watering treatment.

The antimicrobial peptide possesses defence system to virus, fungi and bacteria. To study antibiotic in plant, antimicrobial peptides were obtained by PCR analysis by primers designed from antimicrobial peptides (Gene bank accession no. NM-004345), cloned in pET28 expression vector and the vector transformed into E. coli. And this gene was inserted into Ti-plasmid VB2 vector, which contained the pGD1 promoter. The expression construction was transformed into Agrobacterium EHA105 and then plant tissues of rice (Oryza sativa). Seeds from transgenic plants (T0) were germinated on selective media containing spectinomycin 50 mg/L. Selected plants and wild type were analyzed by PCR and RT-PCR with pGD1 promoter region and transgene specific primer set. All transgenic plants showed expression pattern of similar levels. We showed that the chromobody is effective in binding GFPand antimicrobial peptide gene in tobacco leaf. Most interestingly, this can be applied to interfere with the function of GFP fusion protein and to mislocalize (trap) GFP fusions to the plant cytoplasm in order to alter the phenotype mediated by the targeted proteins. Bacterial blight disease was enhanced resistance in transgenic lines. These results showed that antibiotic peptides might show a broad-spectrum antimicrobial activity.

Korean soybean variety Kwangan was transformed with coat protein (CP), helper component-proteinase (HC-Pro), and ABRE binding factor 3 (ABF3) genes using highly efficient soybean transformation system. Among these genes, CP and HC-Pro were transformed using RNAi technology. Transgenic plants with CP were confirmed for gene introduction and their expression using PCR, real-time PCR, RT-PCR, Southern blot, and Northern blot. To investigate the response of viral infection with CP, T1 plants were inoculated with SMV-infected leaves and confirmed the existence of mosaic symptom in both leaves and seeds. Two transgenic lines with CP were highly resistant to SMV with clear leaves and seeds while SMV-susceptible lines showed mosaic symptom with seed mottling. The transcript levels of T1 plants with CP were also determined by northern blot, suggesting that SMV-resistant T1 plants did not show viral RNA expression whereas SMV-susceptible T1 plants showed viral RNA expression. Currently, the response of viral infection with HC-Pro is investigating to produce SMV-resistant soybean transgenic plants, and the physiological experiment with ABF3 is also carrying out to produce drought-tolerant soybean transgenic plants.

CAX1 유전자가 도입된 형질전환 벼는 T3 세대까지 34계통을 모식물인 일품벼와 공시하였고, 그 외 종자 내 칼슘함량 조사를 비롯한 5가지 실험에서 이전에 실시한 Southern 분석 결과 CAX1 prove의 band 양상이 뚜렷이 나타난 7계 통(T-14, T-15, T-17, T-18, T-19, T-20, T-24)을 T4~T6 계통에서 공시하였다. 앞으로 T7 세대에 대한 특성조사를 거쳐 Ca2+함량이 안정적으로 높게 유지되는 계통을 선발하여 세대육성하면 칼슘과 관련된 유전자의 내재해성 작물의 기초자료 이용뿐만 아니라 농산물의 품질 고급화 및 안정성 향상 그리고 병충해에 의한 농작물 피해를 줄일 수 있는 효과를 가져 오고자 수행한 결과는 다음과 같다. T4 세대에서 형질이 안정적으로 발현되면서 모품종과 유사하고 농업적 형질이 우수한 5 계통을 선발하여 T6 세대까지 농업적인 생육 안정성(수장, 수수, 간장)은 15, 17, 18, 24 계통들은 출수기가 모품종인 일품벼와 비슷한 경향이었고 14, 19, 20계통들은 일품벼보다 6~11일까지 늦어지는 경향이었다. 형질전환체 7 계통의 간장을 조사한 결과 14 계통을 제외하고는 대부분 모품종인 일품벼와 유사하게 나타났다. CAX1 형질전환체인 T5~T6 세대에서 병저항성은 일품 모품종보다 벼도열병(blast)과 벼잎집무늬마름병(sheath blight)의 방제가가 낮게 나왔으며, 벼도열병은 2 년간 모두 모품종에 대비해 T-14, 17, 18, 19, 20은 방제가가 높게 나타나 병저항성이 있는 것으로 나타났다. 그러나 벼잎집무늬마름병은 T-14, 19, 20, 24 계통이 방제가가 높게 나타났으며, 벼도열병과 벼잎집무늬마름병 모두 방제가가 높은 계통은 T-14, 19, 20 으로 조사되었다. 저온처리 된 벼의 초장 조사에서 각각 온실과 17℃ 의 두 조건에 있어서 온실에서의 초장 조사와 생존 개체수를 대비해 볼 때 일품벼와 형질전환체 모두 정상적인 발육상태를 보였으나, 저온조건에서 초장 조사는 일품벼에 비해 T6 -19을 제외한 형질전환 계통들이 양호하였으며 T6 -18, 24 계통은 내한성 품종인 상주벼와 유사하거나 조금 차이가 났다. 종자내의 캄슘함량을 분석하기 위하여 T3~T7 종자를 이용하여 얻어진 결과는 T3 종자에서 452.2~1,376.2%로 칼슘함량이 증가되었고 분자 marker에 의해 선발하여 된 T7종자까지 분석한바 13.4~68.0%로 나타남을 확인할 수 있었다.